Tert-butyl N-[2-{4-[6-amino-5-(2,4-difluorobenzoyl)-2-oxopyridin-1(2H)-yl]-3,5-difluorophenyl}ethyl]-L-alaninate or a salt,hydrate or solvate thereof

ABSTRACT

The present invention provides a compound which is: tert-butyl N-[2-{4-[6-amino-5-(2,4-difluorobenzoyl)-2-oxopyridin-1(2H)-yl]-3,5-difluorophenyl}ethyl)-L-alaninate or a salt, hydrate or solvate thereof. The present invention also provides a pharmaceutical composition comprising the compound together with one or more pharmaceutically acceptable carriers and/or excipients. The compound and composition are useful for inhibiting the activity of a p38 MAP kinase enzyme. As such they may be used in the treatment of a autoimmune or inflammatory disease, or a cell proliferative disease. In addition, the invention provides an acid produced by hydrolysis of the ester group of the compound of the invention. The acid is N-[2-{4-[6-amino-5-(2,4-difluorobenzoyl)-2-oxopyridin-1(2H)-yl]-3,5-difluorophenyl}ethyl)-L-alanine.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.14/436,404 filed Apr. 16, 2015, which is a national phase applicationunder 35 U.S.C. § 371 that claims priority to International ApplicationNo. PCT/GB2013/052689 filed Oct. 15, 2013, which claims the benefit ofpriority to Great Britain Patent Application Nos. 1306881.2 filed onApr. 16, 2013 and 1218640.9 filed on Oct. 17, 2012, all of which areincorporated herein by reference in their entirety.

FIELD OF THE INVENTION

The invention relates to an amino acid ester compound and tocompositions comprising the amino acid ester compound. The inventionalso relates to use of the compound or composition in the inhibition ofthe p38 MAP kinase enzyme. In addition, the invention relates to an acidproduced by hydrolysis of the ester group of the compound of theinvention.

BACKGROUND OF THE INVENTION

Inappropriate activation of leukocytes including monocytes, macrophagesand neutrophils leading to the production of elevated levels ofcytokines such as TNF-α, IL1-β and IL-8, is a feature of thepathogenesis of several inflammatory diseases including rheumatoidarthritis, ulcerative colitis, Crohn's disease, chronic obstructivepulmonary disease (COPD), asthma and psoriasis, and cell proliferativediseases with an inflammatory component. The production of cytokines byinflammatory cells is a result of response to a variety of externalstimuli, leading to the activation of a number of intracellularsignalling mechanisms. Prominent amongst these is the mitogen-activatedprotein kinase (MAPK) superfamily consisting of highly conservedsignalling kinases that regulate cell growth, differentiation and stressresponses. Mammalian cells contain at least three families of MAPKs: thep42/44 extracellular signal-regulated kinase (ERK) MAPKs, c-JunNH2-terminal kinases (JNKs) and p38 MAPK (also termedp38a/Mpk2/RK/SAPK2a/CSBP1/2). p38 MAPK was first cloned following itsidentification as a kinase that is tyrosine phosphorylated afterstimulation of monocytes by lipopolysaccharide (LPS) [Han et al, Science1994, 265, 808]. Additional homologues of mammalian p38 have beendescribed and include p38β [Jiang et al, J. Biol. Chem, 1996, 271,17920], p38γ [Li et al, Biochem. Biophys. Res. Commun., 1996, 228, 334]and p38 γ [Jiang et al, J.Biol.Chem. 1997, 272, 30122]. While p38α andp38 β are ubiquitously expressed, p38 γ is restricted primarily toskeletal muscle and p38 δ is predominantly expressed in lung and kidney.

The release of cytokines by host defense cells and the response ofleukocytes to cytokines and other pro-inflammatory stresses are tovarying extent regulated by p38 MAPK [Cuenda et al, FEBS Lett, 1995,364, 229-233]. In other cell types, p38 MAPK controls stress responsessuch as the production of IL-8 by bronchial epithelial cells stimulatedby TNF-α and the up-regulation of the cell adhesion molecule ICAM-1 inLPS-stimulated endothelial cells. Upon activation, via dualphosphorylation of a TGY motif by the dual specificity kinases MKK3 andMKK6, p38 MAPK exerts its effects through phosphorylation oftranscription factors and other kinases. MAP kinase-activated proteinkinase-2 (MAPKAP-K2) has been identified as a target for p38phosphorylation. It has been demonstrated that mice [Kotlyarov et al,Nat. Cell Biol. 1999, 1, 94-97] lacking MAPKAP-K2 release reduced levelsof TNF-α, IL-1β, IL-6, IL-10 and IFN-γ in response to LPS/galactosaminemediated endotoxic shock. The regulation of the levels of thesecytokines as well as COX-2 is at the mRNA level. TNF-α levels areregulated through translational control via AU-rich elements of the3′-UTR of TNF-α mRNA, with MAPKAP-K2 signalling increasing TNF-α mRNAtranslation. MAPKAP-K2 signalling leads to increased mRNA stability forCOX-2, IL-6 and macrophage inflammatory protein. MAPKAP-K2 determinesthe cellular location of p38 MAPK as well as transducing p38 MAPKsignalling, possessing a nuclear localisation signal at its carboxylterminus and a nuclear export signal as part of its autoinhibitorydomain [Engel et al, EMBO J. 1998, 17, 3363-3371]. In stressed cells,MAPKAP-K2 and p38 MAPK migrate to the cytoplasm from the nucleus, thismigration only occurring when p38 MAPK is catalytically active. It isbelieved that this event is driven by the exposure of the MAPKAP-K2nuclear export signal, as a result of phosphorylation by p38 MAPK [Menget al, J. Biol. Chem. 2002, 277, 37401-37405]. Additionally p38 MAPKeither directly or indirectly leads to the phosphorylation of severaltranscription factors believed to mediate inflammation, including ATF1/2 (activating transcription factors 1/2), CHOP-10/GADD-153 (growtharrest and DNA damage inducible gene 153), SAP-1 (serum response factoraccessory protein-1) and MEF2C (myocyte enhancer factor-2) [Foster etal, Drug News Perspect. 2000, 13, 488-497].

It has been demonstrated in several instances that the inhibition of p38MAPK activity by small molecules, is useful for the treatment of severaldisease states mediated by inappropriate cytokine production includingrheumatoid arthritis, COPD, asthma and cerebral ischemia. This modalityhas been the subject of several reviews [Salituro et al, CurrentMedicinal Chemistry, 1999, 6, 807-823 and Kumar et al, Nature ReviewsDrug Discovery 2003, 2, 717-726].

Inhibitors of p38 MAPK have been shown to be efficacious in animalmodels of rheumatoid arthritis, such as collagen-induced arthritis inrat [Revesz et al, Biorg. Med. Chem. Lett., 2000, 10, 1261-1364] andadjuvant-induced arthritis in rat [Wadsworth et al, J. Pharmacol. Exp.Ther., 1999, 291, 1685-1691]. In murine models of pancreatitis-inducedlung injury, pretreatment with a p38 MAPK inhibitor reduced TNF-αrelease in the airways and pulmonary edema [Denham et al, Crit. CareMed., 2000, 29, 628 and Yang et al, Surgery, 1999, 126, 216]. Inhibitionof p38 MAPK before ovalbumin (OVA) challenge in OVA-sensitized micedecreased cytokine and inflammatory cell accumulation in the airways inan allergic airway model of inflammation [Underwood et al, J. Pharmacol.Exp. Ther., 2000, 293, 281]. Increased activity of p38 MAP kinase hasbeen observed in patients suffering from inflammatory bowel disease[Waetzig et al, J. Immunol, 2002, 168, 5432-5351]. p38 MAPK inhibitorshave been shown to be efficacious in rat models of cardiac hypertrophy[Behr et al, Circulation, 2001, 104, 1292-1298] and cerebral focalischemia [Barone et al, J. Pharmacol. Exp. Ther., 2001, 296, 312-321].

WO 2007/129040 and WO 2009/060160 both disclose alpha amino acid estersthat are inhibitors of p38 MAP kinase. In the compounds disclosed, theester group is an ester group which is hydrolysable by one or moreintracellular esterase enzymes to a carboxylic acid group; and thesubstituents on the carbon of the alpha amino acid ester form a sidechain, which is the side chain of a natural or non-natural alpha aminoacid.

The compounds disclosed are stated to be potent and selective inhibitorsof p38 MAPK (p38α, β, γ, and δ) and the isoforms and splice variantsthereof especially p38α, p38β2 and p38β2. The compounds are thus of usein medicine, for example in the treatment and prophylaxis of immune andinflammatory disorders described herein. The compounds are characterisedby the presence in the molecule of the amino acid motif or amino acidester motif —NH—CHR1R2 which is hydrolysable by an intracellularcarboxylesterase. The compounds having the lipophilic amino acid estermotif cross the cell membrane, and are hydrolysed to the acid by theintracellular carboxylesterases. The polar hydrolysis productaccumulates in the cell since it does not readily cross the cellmembrane. Hence the p38 MAP kinase activity of the compounds areprolonged and enhanced within the cell. The compounds of that inventionare related to the p38 MAP kinase inhibitors encompassed by thedisclosures in International Patent Application WO03076405 but differ inthat they have the amino acid ester motif referred to above.

WO 2007/129040 also disclosed that the compounds with which it isconcerned include those which selectively accumulate in macrophages.Macrophages are known to play a key role in inflammatory disordersthrough the release of cytokines in particular TNFα and IL-1 (van Roonet al, Arthritis and Rheumatism, 2003, 1229-1238). In rheumatoidarthritis they are major contributors to the maintenance of jointinflammation and joint destruction. Macrophages are also involved intumour growth and development (Naldini and Carraro, Curr Drug TargetsInflamm Allergy, 2005, 3-8). Hence agents that selectively targetmacrophage cell proliferation could be of value in the treatment ofcancer and autoimmune disease. Targeting specific cell types would beexpected to lead to reduced side-effects. The way in which the esterasemotif is linked to the p38 kinase inhibitor determines whether it ishydrolysed, and hence whether or not it accumulates in different celltypes. Specifically, macrophages contain the human carboxylesterasehCE-1 whereas other cell types do not. In the general formula (I) of WO2007/129040, when the nitrogen of the esterase motif R1CH(R2)NH— is notdirectly linked to a carbonyl (—C(═O)—), ie when Y is not a —C(═O),—C(═O)O— or —C(═O)NR3-radical, the ester will only be hydrolysed byhCE-1 and hence the inhibitors will only accumulate in macrophages.Herein, unless “monocyte” or “monocytes” is specified, the termmacrophage or macrophages will be used to denote macrophages (includingtumour associated macrophages) and/or monocytes.

WO 2009/060160 discloses a group of specific compounds falling withinthe general disclosures of WO 2007/129040, but not specificallyidentified or exemplified therein. The compounds display the macrophageselectivity property discussed above.

SUMMARY OF THE INVENTION

The present inventors have found that the compound tert-butylN-[2-{4-[6-amino-5-(2,4-difluorobenzoyl)-2-oxopyridin-1(2H)-yl]-3,5-difluorophenyl}ethyl)-L-alaninateis surprisingly good at inhibiting p38 MAP kinase activity. Tests haveshown that the IC50 value for ester TNF-α inhibition in human blood issignificantly lower than would be expected given the IC50 values forester TNF-α inhibition in human blood observed for related compounds.Further, tests carried out by the inventors have demonstrated that thebioavailability of the compound of the invention is much higher thanwould have been predicted from structurally-similar, known compounds.

Accordingly, the present invention provides a compound which is:tert-butylN-[2-{4-[6-amino-5-(2,4-difluorobenzoyl)-2-oxopyridin-1(2H)-yl]-3,5-difluorophenyl}ethyl)-L-alaninateor a pharmaceutically acceptable salt, hydrate or solvate thereof.

The present invention also provides a pharmaceutical compositioncomprising the compound together with one or more pharmaceuticallyacceptable carriers and/or excipients.

In another aspect, the invention provides a compound as defined above ora composition as defined above for use in a method of treatment of thehuman or animal body by therapy.

The present invention also provides a compound as defined above or acomposition as defined above for use in the inhibition of the activityof a p38 MAP kinase enzyme in vitro or in vivo. There is furtherprovided a compound as defined above or a composition as defined abovefor use in the prevention or treatment of autoimmune or inflammatorydisease. Also provided is a compound as defined above or a compositionas defined above for use in the treatment of a cell proliferativedisease.

In another aspect, the invention provides a method of inhibiting theactivity of a p38 MAP kinase enzyme, which method comprises contactingthe enzyme with an amount of a compound as defined above or acomposition as defined above effective for such inhibition. Alsoprovided is a method of treating or preventing autoimmune orinflammatory disease in a subject, which method comprises administeringto said subject an effective amount of a compound as defined above or acomposition as defined above. There is further provided a method oftreating a cell proliferative disease in a subject, which methodcomprises administering to said subject an effective amount of acompound as defined above or a composition as defined above. Saidtreatment may comprise ameliorating or reducing the incidence of thecell proliferative disease.

In yet another aspect, the invention provides the use of a compound asdefined above or a composition as defined above in the manufacture of amedicament for inhibiting the activity of a p38 MAP kinase enzyme.Further provided is the use of a compound as defined above or acomposition as defined above in the manufacture of a medicament for theprevention or treatment of an autoimmune or inflammatory disease. Alsoprovided is the use of a compound as defined above or a composition asdefined above in the manufacture of a medicament for the treatment of acell proliferative disease.

The invention also provides an agent for inhibiting the activity of ap38 MAP kinase enzyme comprising a compound as defined above or acomposition as defined above as active ingredient. The agent istypically for the prevention or treatment of autoimmune or inflammatorydisease. It may alternatively be for the treatment of cell proliferativedisease.

Also provided by the invention is an acid which is:N-[2-{4-[6-amino-5-(2,4-difluorobenzoyl)-2-oxopyridin-1(2H)-yl]-3,5-difluorophenyl}ethyl)-L-alanine.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a compound which is: tert-butylN-[2-{4-[6-amino-5-(2,4-difluorobenzoyl)-2-oxopyridin-1(2H)-yl]-3,5-difluorophenyl}ethyl)-L-alaninate.

The compound of the invention may be prepared in the form of a salt,hydrate or solvate. The invention thus also provides a salt, hydrate orsolvate of the compound. Typically, the salt is a pharmaceuticallyacceptable salt.

As used herein, a pharmaceutically acceptable salt is a salt with apharmaceutically acceptable acid or base. Pharmaceutically acceptableacids include both inorganic acids such as hydrochloric, sulphuric,phosphoric, diphosphoric, hydrobromic or nitric acid and organic acidssuch as citric, salicylic, glutamic, lactic, fumaric, maleic, malic,ascorbic, succinic, tartaric, benzoic, acetic, methanesulphonic,ethanesulphonic, benzenesulphonic or p-toluenesulphonic acid.Pharmaceutically acceptable bases include alkali metal (e.g. sodium orpotassium) and alkali earth metal (e.g. calcium, barium or magnesium)hydroxides and organic bases such as alkyl amines, aralkyl amines andheterocyclic amines. Examples of suitable organic bases include, but arenot limited to, N-methyl-D-glucamine, cholinetris(hydroxymethyl)amino-methane, L-arginine, L-lysine, N-ethylpiperidine, dibenzylamine. For a review on suitable salts, see Handbookof Pharmaceutical Salts: Properties, Selection, and Use by Stahl andWermuth (Wiley-VCH, Weinheim, Germany, 2002).

Suitable salts of the compounds of the invention include those mentionedherein as examples of pharmaceutically acceptable salts.

The term ‘solvate’ is used herein to describe a molecular complexcomprising the compound of the invention and a stoichiometric amount ofone or more pharmaceutically acceptable solvent molecules, for example,ethanol. The term ‘hydrate’ is employed when said solvent is water.

For the avoidance of doubt, the compound of the invention may be used inany tautomeric form.

The compound of the invention includes a chiral centre. The compound istypically in the form of the L-alaninate derivative (i.e. as depicted inExample 1). However, the compound may exist as the D-alaninatederivative or as a mixture of the D- and L-forms. Where a mixture ispresent, preferably at least 90%, 95% or 99% is present as theL-alaninate derivative.

A suitable scheme and process for the production of the compound of theinvention, with reference to the examples section which follows, isdiscussed below.

The starting materials are typically 4-Chlorophenyl3-(2,4-difluorophenyl)-3-oxopropanimidothioate hydro-chloride and2-(4-Amino-3,5-difluorophenyl)ethanol.2-(4-Amino-3,5-difluorophenyl)ethanol may be prepared using thefollowing scheme, which is analogous to scheme 1 of the examplessection:

Difluoronitrobenzene is commercially available. Stage 1 requires theaddition of a tert-butyl acetate group to the phenyl ring, para to thenitro group. Stage 2 requires the hydrolysis of the ester group to formthe corresponding acid. The acid is reduced to a primary alcohol instage 3. In stage 4 the nitro group is reduced to an amine.

4-Chlorophenyl 3-(2,4-difluorophenyl)-3-oxopropanimidothioatehydro-chloride may be prepared using experimental procedures describedin WO 2003076405.

The compound, tert-ButylN-(2-{4-[6-amino-5-(2,4-difluorobenzoyl)-2-oxopyridin-1(2H)-yl]-3,5-difluorophenyl}ethyl)-L-alaninatemay then be synthesised using the following scheme, which is analogousto scheme 2 of the examples section.

In stage 1, the 2-(4-Amino-3,5-difluorophenyl)ethanol and 4-Chlorophenyl3-(2,4-difluorophenyl)-3-oxopropanimidothioate hydro-chloride arereacted together to form2-(4-{[3-(2,4-Difluorophenyl)-3-oxopropanimidoyl]amino}-3,5-difluorophenyl)ethylacetate. In stage 2, propiolic acid is added to form2-{4-[6-Amino-5-(2,4-difluorobenzoyl)-2-oxopyridin-1(2H)-yl]-3,5-difluorophenyl}ethylacetate. In stage 3, the acetate group is hydrolysed to leave an alcoholand in stage 4 the resulting alcohol group is oxidised to an aldehyde.The compound of the invention is then formed in stage 5, by the additionof tert-butyl L-alaninate hydrochloride. Tert-butyl L-alaninatehydrochloride is commercially available.

The invention also provides a pharmaceutical composition comprising thecompound together with one or more pharmaceutically acceptable carriersand/or excipients. Said pharmaceutical composition typically contains upto 85 wt % of a compound of the invention. More typically, it containsup to 50 wt % of a compound of the invention. Preferred pharmaceuticalcompositions are sterile and pyrogen free.

The compound of the invention may be administered in a variety of dosageforms. Thus, they can be administered orally, for example as tablets,troches, capsules, lozenges, aqueous or oily suspensions, dispersiblepowders or granules. The compounds of the invention may also beadministered parenterally, either subcutaneously, intravenously,intramuscularly, intrasternally, transdermally or by infusiontechniques. Depending on the vehicle and concentration used, the drugcan either be suspended or dissolved in the vehicle. Advantageously,adjuvants such as a local anaesthetic, preservative and buffering agentcan be dissolved in the vehicle. The compounds may also be administeredas suppositories. The compounds may be administered by inhalation in theform of an aerosol via an inhaler or nebuliser.

A compound of the invention is typically formulated for administrationwith a pharmaceutically acceptable carrier or diluent. For example,solid oral forms may contain, together with the active compound,solubilising agents, e.g. cyclodextrins or modified cyclodextrins;diluents, e.g. lactose, dextrose, saccharose, cellulose, corn starch orpotato starch; lubricants, e.g. silica, talc, stearic acid, magnesium orcalcium stearate, and/or polyethylene glycols; binding agents; e.g.starches, arabic gums, tragacanth gums, gelatin, syrup, acacia,sorbitol, methylcellulose, carboxymethylcellulose or polyvinylpyrrolidone; disaggregating agents, e.g. starch, alginic acid, alginatesor sodium starch glycolate; effervescing mixtures; dyestuffs;sweeteners; wetting agents, such as lecithin, polysorbates,laurylsulphates; and, in general, non-toxic and pharmacologicallyinactive substances used in pharmaceutical formulations. Suchpharmaceutical preparations may be manufactured in a known manner, forexample, by means of mixing, granulating, tabletting, sugar-coating, orfilm coating processes.

Liquid dispersions for oral administration may be solutions, syrups,emulsions and suspensions. Liquid preparations may contain conventionaladditives such as suspending agents, for example sorbitol, syrup, methylcellulose, glucose syrup, gelatin hydrogenated edible fats; emulsifyingagents, for example lecithin, sorbitan monooleate, or acacia;non-aqueous vehicles (which may include edible oils), for example almondoil, fractionated coconut oil, oily esters such as glycerine, propyleneglycol, or ethyl alcohol; preservatives, for example methyl or propylp-hydroxybenzoate or sorbic acid, and if desired conventional flavouringor colouring agents. The solutions may contain solubilising agents e.g.cyclodextrins or modified cyclodextrins. The syrups may contain ascarriers, for example, saccharose or saccharose with glycerine and/ormannitol and/or sorbitol.

Suspensions and emulsions may contain as carrier, for example a naturalgum, agar, sodium alginate, pectin, methylcellulose,carboxymethylcellulose, or polyvinyl alcohol. The suspensions orsolutions for intramuscular injections may contain, together with theactive compound, a pharmaceutically acceptable carrier, e.g. sterilewater, olive oil, ethyl oleate, glycols, e.g. propylene glycol;solubilising agents, e.g. cyclodextrins or modified cyclodextrins, andif desired, a suitable amount of lidocaine hydrochloride.

Solutions for intravenous or infusions may contain as carrier, forexample, sterile water and solubilising agents, e.g. cyclodextrins ormodified cyclodextrins or preferably they may be in the form of sterile,aqueous, isotonic saline solutions.

For topical application to the skin, the drug may be made up into acream, lotion or ointment. Cream or ointment formulations which may beused for the drug are conventional formulations well known in the art,for example as described in standard textbooks of pharmaceutics such asthe British Pharmacopoeia.

For topical application by inhalation, the drug may be formulated foraerosol delivery for example, by pressure-driven jet atomizers orultrasonic atomizers, or preferably by propellant-driven meteredaerosols or propellant-free administration of micronized powders, forexample, inhalation capsules or other “dry powder” delivery systems.Excipients, such as, for example, propellants (e.g. Frigen in the caseof metered aerosols), surface-active substances, emulsifiers,stabilizers, preservatives, flavorings, and fillers (e.g. lactose in thecase of powder inhalers) may be present in such inhaled formulations.For the purposes of inhalation, a large number of apparata are availablewith which aerosols of optimum particle size can be generated andadministered, using an inhalation technique which is appropriate for thepatient. In addition to the use of adaptors (spacers, expanders) andpear-shaped containers (e.g. Nebulator®, Volumatic®), and automaticdevices emitting a puffer spray (Autohaler®), for metered aerosols, inparticular in the case of powder inhalers, a number of technicalsolutions are available (e.g. Diskhaler®, Rotadisk®, Turbohaler® or theinhalers for example as described in European Patent Application EP 0505 321).

For topical application to the eye, the drug may be made up into asolution or suspension in a suitable sterile aqueous or non aqueousvehicle. Additives, for instance buffers such as sodium metabisulphiteor disodium edeate; preservatives including bactericidal and fungicidalagents such as phenyl mercuric acetate or nitrate, benzalkonium chlorideor chlorhexidine, and thickening agents such as hypromellose may also beincluded.

A therapeutically effective amount of a compound of the invention isadministered to a subject. It will be understood that the specific doselevel for any particular subject will depend upon a variety of factorsincluding the activity of the specific compound employed, the age, bodyweight, general health, sex, diet, time of administration, route ofadministration, rate of excretion, drug combination and the severity ofthe particular disease undergoing treatment. Optimum dose levels andfrequency of dosing will usually be determined by clinical trial.

A typical daily dose is up to 50 mg per kg of body weight, for examplefrom 0.001 to 50 mg per kg of body weight, according to the activity ofthe specific compound, the age, weight and conditions of the subject tobe treated, the type and severity of the disease and the frequency androute of administration. Preferably, daily dosage levels are from 0.05mg to 2 g, preferably from 0.1 mg to 10 mg. The compound of theinvention is typically administered to the patient in a non-toxicamount.

The invention also provides a compound as defined herein or acomposition as defined herein for use in a method of treatment of thehuman or animal body by therapy.

The compounds and compositions of the invention have been found toinhibit the activity of a p38 MAP kinase enzyme. The compounds andcompositions are therefore useful in the prevention and treatment ofdiseases and conditions modulated by p38 MAP kinase activity. Diseasesand conditions modulated by p38 MAP kinase activity include cellproliferative disease such as cancer and psoriasis, polyglutaminedisease such as Huntingdon's disease, neurodegenerative disease such asAlzheimers disease, autoimmune disease such as rheumatoid arthritis,diabetes, haematological disease, inflammatory disease, cardiovasculardisease, atherosclerosis, and the inflammatory sequelia of infection.Particular examples are cell proliferative disease, autoimmune diseaseand inflammatory disease.

Autoimmune disease often has an inflammatory component. Such conditionsinclude acute disseminated alopecia universalise, ANCA positivediseases, Behcet's disease, Chagas' disease, chronic fatigue syndrome,dysautonomia, encephalomyelitis, ankylosing spondylitis, aplasticanemia, hidradenitis suppurativa, autoimmune hepatitis, autoimmuneoophoritis, celiac disease, inflammatory bowel disease, Crohn's disease,diabetes mellitus type 1, Fanconi syndrome, giant cell arteritis,glomerulonephritis, Goodpasture's syndrome, Grave's disease,Guillain-Barre syndrome, Hashimoto's disease, Henoch-Schönlein purpura,Kawasaki's disease, systemic lupus erythematosus, microscopic colitis,microscopic polyarteritis, mixed connective tissue disease, multiplesclerosis, myasthenia gravis, opsocionus myoclonus syndrome, opticneuritis, Ord's thyroiditis, pemphigus, polyarteritis nodosa,polymyalgia, rheumatoid arthritis, Reiter's syndrome, Sjogren'ssyndrome, temporal arteritis, Wegener's granulomatosis, warm autoimmunehaemolytic anemia, interstitial cystitis, lyme disease, morphea,psoriasis, sarcoidosis, scleroderma, ulcerative colitis, and vitiligo.

Other inflammatory conditions which may be prevented or treated with thecompounds and compositions of the invention include, for example,appendicitis, dermatitis, dermatomyositis, endocarditis, fibrositis,gingivitis, glossitis, hepatitis, hidradenitis suppurativa, iritis,laryngitis, mastitis, myocarditis, nephritis, otitis, pancreatitis,parotitis, percarditis, peritonoitis, pharyngitis, pleuritis,pneumonitis, prostatistis, pyelonephritis, and stomatisi, transplantrejection (involving organs such as kidney, liver, heart, lung, pancreas(e.g., islet cells), bone marrow, cornea, small bowel, skin allografts,skin homografts, and heart valve xengrafts, sewrum sickness, and graftvs host disease), acute pancreatitis, chronic pancreatitis, acuterespiratory distress syndrome, Sexary's syndrome, congenital adrenalhyperplasis, nonsuppurative thyroiditis, hypercalcemia associated withcancer, pemphigus, bullous dermatitis herpetiformis, severe erythemamultiforme, exfoliative dermatitis, seborrheic dermatitis, seasonal orperennial allergic rhinitis, bronchial asthma, contact dermatitis,astopic dermatitis, drug hypersensistivity reactions, allergicconjunctivitis, keratitis, herpes zoster ophthalmicus, iritis andoiridocyclitis, chorioretinitis, optic neuritis, symptomaticsarcoidosis, fulminating or disseminated pulmonary tuberculosischemotherapy, idiopathic thrombocytopenic purpura in adults, secondarythrombocytopenia in adults, acquired (autoimmune) haemolytic anemia,leukaemia and lymphomas in adults, acute leukaemia of childhood,regional enteritis, autoimmune vasculitis, multiple sclerosis, chronicobstructive pulmonary disease, solid organ transplant rejection, sepsis,primary biliary cirrhosis and primary sclerosing cholangitis.

The compounds and compositions of the invention are useful in theprevention and treatment of inflammatory and autoimmune diseases andconditions. Inflammatory and autoimmune diseases and conditions whichcan be treated using the compounds and compositions of the inventioninclude rheumatoid arthritis, psoriatic arthritis, Type 1 diabetes,asthma, inflammatory bowel disease, systemic lupus erythematosis, andinflammation accompanying infectious conditions (e.g., sepsis),psoriasis, Crohns disease, ulcerative colitis, chronic obstructivepulmonary disease, multiple sclerosis, atopic dermatitis, and graftversus host disease.

The compounds and compositions of the invention are also useful in thetreatment of a cell proliferative disease, for example cancer. Examplesof cancers which can be treated include breast cancer, ovarian cancer,lung cancer, pancreatic cancer, hepatic cancer, colon cancer, renalcancer, lymphoma and melanoma. For instance, cancers which may betreated include breast cancer, ovarian cancer, pancreatic cancer, lungcancer, colon cancer, renal cancer, lymphoma or melanoma.

The invention also relates to an acid which is:

N-[2-{4-[6-amino-5-(2,4-difluorobenzoyl)-2-oxopyridin-1(2H)-yl]-3,5-difluorophenyl}ethyl)-L-alanine

The acid may be prepared by hydrolysis of the compound of the invention.

As discussed herein, the compound of the invention is macrophageselective. Thus, the ester group of the compound of the invention ishydrolysable by cells containing the human carboxylesterase hCE-1 andnot by cells containing hCE-2 or hCE-3. The acid is therefore producedwithin the cells containing hCE-1 on hydrolysis of the ester group ofthe compound of the invention and the acid selectively accumulateswithin such cells.

The present invention is further illustrated in the Examples whichfollow.

EXAMPLES

The compound of the invention may be prepared according to the followingExample.

Abbreviations

CDI=carbonyldiimidazole

DCM=dichloromethane

DMF=dimethylformamide

EtOAc=ethyl acetate

HCl=hydrochloric acid

LCMS=high performance liquid chromatography/mass spectrometry

MeOH=methanol

MgSO4=magnesium sulphate

Na₂CO₃=sodium carbonate

NaHCO₃=sodium hydrogen carbonate

NMR=nuclear magnetic resonance

STAB=sodium triacetoxyborohydride

THF=tetrahydrofuran

g=gram(s)

mg=milligram(s)

mL=millilitre(s)

mmol=millimole(s)

Commercially available reagents and solvents (HPLC grade) were usedwithout further purification. Solvents were removed using a Buchi rotaryevaporator. Microwave irradiation was carried out using a BiotageInitiator™ Eight microwave synthesiser. Purification of compounds byflash chromatography column was performed using silica gel, particlesize 40-63 μm (230-400 mesh) obtained from Fluorochem.

¹H NMR spectra were recorded on a Bruker 300 MHz AV spectrometer indeuterated solvents. Chemical shifts (d) are in parts per million.Thin-layer chromatography (TLC) analysis was performed with Kieselgel 60F₂₅₄ (Merck) plates and visualized using UV light.

Analytical HPLC/MS was performed on an Agilent HP1100 LC system usingreverse phase Luna C18 columns (3 mm, 50×4.6 mm), gradient 5-95% B(A=water/0.1% Formic acid, B=acetonitrile/0.1% Formic acid) over 2.25min, flow=2.25 mL/min. UV spectra were recorded at 220 and 254 nm usinga G1315B DAD detector. Mass spectra were obtained over the range m/z 150to 800 on a LC/MSD SL G1956B detector. Data were integrated and reportedusing ChemStation and ChemStation Data Browser software.

Intermediates Intermediate 1: 4-Chlorophenyl3-(2,4-difluorophenyl)-3-oxopropanimidothioate hydro-chloride

Intermediate 1 can be prepared using experimental procedures describedin WO 2003076405.

Intermediate 2: 2-(4-Amino-3,5-difluorophenyl)ethanol

Intermediate 2 was synthesised using the route shown in Scheme 1 below.

Stage 1—tert-Butyl(3,5-difluoro-4-nitrophenyl)acetate

A solution of difluoronitrobenzene (24.96 g, 157 mmol) and tert-butylchloroacetate (38.0 mL, 267 mmol) in anhydrous DMF (200 mL) was addeddropwise over one hour to a cold (−35° C.) suspension of potassiumtert-butoxide (61.61 g, 549 mmol) in anhydrous DMF (200 mL) undernitrogen. The reaction mixture was stirred at −35° C. for 1.5 hours,quenched with 2N HCl (240 mL) and extracted with heptanes (4×200 mL).The combined organic extracts were washed with water (3×200 mL), brine(200 mL), dried (MgSO₄), filtered and concentrated under reducedpressure to leave a yellow oil. Purification by column chromatography(10% EtOAc in heptanes) afforded a yellow oil (37.64 g). Another twobatches (10.00 g and 23.54 g of difluoronitrobenzene) afforded 14.30 gand 31.39 g of product respectively. ¹H NMR's of all three batchesshowed a mixture of desired compound and small amounts of unidentifiedimpurities. The 3 batches were combined and used in the next stagewithout further purification.

¹H NMR (300 MHz, CDCl₃) 7.05 (2H, d, J=8.5 Hz), 3.56 (2H, s), 1.46 (9H,s).

Stage 2—(3,5-Difluoro-4-nitrophenyl)acetic acid

Trifluoroacetic acid (150 mL) was added dropwise over 20 minutes to acold (0° C.) solution of tert-butyl (3,5-difluoro-4-nitrophenyl)acetate(83.33 g, 305 mmol) in DCM (300 mL). On completion of the addition, thereaction mixture was allowed to warm to room temperature and stirred for3 hours. The reaction mixture was concentrated under reduced pressure toleave a sticky brown solid. Trituration with heptanes afforded the titlecompound as a yellow solid (53.29 g, 67% yield over two steps).

¹H NMR (300 MHz, CDCl₃) 7.08 (2H, d, J=8.5 Hz), 3.74 (2H, s), —CO₂ H notvisible.

Stage 3—2-(3,5-Difluoro-4-nitrophenyl)ethanol

Borane-dimethyl sulfide complex (35 mL, 368 mmol) was added dropwiseover 20 minutes to a cold (0° C.) solution of(3,5-difluoro-4-nitrophenyl)acetic acid (53.29 g, 245 mmol) in anhydrousTHF (500 mL) under nitrogen. Upon completion of the addition, thereaction mixture was allowed to warm to room temperature, stirred for 16hours, cooled to 0° C., carefully quenched with MeOH (300 mL) andconcentrated under reduced pressure to leave a brown oil. Purificationby dry flash chromatography (60-80% EtOAc in heptanes) afforded thetitle compound as an orange oil (38.90 g, 78% yield).

¹H NMR (300 MHz, CDCl₃) 7.01 (2H, d, J=8.7 Hz), 3.93 (2H, t, J=6.2 Hz),2.92 (2H, t, J=6.2 Hz), 2.34 (1H, br s).

Stage 4—2-(4-Amino-3,5-difluorophenyl)ethanol

2-(3,5-Difluoro-4-nitrophenyl)ethanol (38.90 g, 191 mmol) was dissolvedin EtOAc (250 mL). The reaction vessel was evacuated and filled withnitrogen three times. Palladium on carbon (10 wt %, 4.00 g) was addedand the vessel was evacuated and filled with nitrogen three times.Finally, the vessel was evacuated and filled with hydrogen and fittedwith a balloon containing hydrogen. After, stirring at room temperatureunder hydrogen for 15 hours, the hydrogen balloon was refilled and themixture stirred for an additional 25 hours. The reaction mixture wasfiltered through Celite® and the filtrate was concentrated under reducedpressure to leave a brown oil. Purification by dry flash chromatography(50% EtOAc in heptanes) afforded the title compound as a beige solid(20.70 g, 62% yield).

¹H NMR (300 MHz, CDCl₃) 6.73-6.70 (2H, m), 3.81 (2H, t, J=6.4 Hz), 2.75(2H, t, J=6.4 Hz), —OH and —NH ₂ not visible.

Example 1 tert-ButylN-(2-{4-[6-amino-5-(2,4-difluorobenzoyl)-2-oxopyridin-1(2H)-yl]-3,5-difluorophenyl}ethyl)-L-alaninate

Example 1 was synthesised using the route shown in Scheme 2 below.

Stage1—2-(4-{[3-(2,4-Difluorophenyl)-3-oxopropanimidoyl]amino}-3,5-difluorophenyl)ethylacetate

2-(4-Amino-3,5-difluorophenyl)ethanol (20.71 g, 120 mmol) was added to asolution of 4-chlorophenyl3-(2,4-difluorophenyl)-3-oxopropanimidothioate hydrochloride (41.26 g,114 mmol) in glacial acetic acid (400 mL). The reaction mixture wasstirred at 80° C. for 2.5 hours and acetic anhydride (21 mL, 228 mmol)was added. After an additional 45 minutes at 80° C., the reactionmixture was allowed to cool to room temperature and concentrated underreduced pressure to leave a brown oil. Trituration with EtOAc afforded abeige solid, which was washed with diethyl ether. The solid was taken upin a saturated aqueous solution of NaHCO₃ and vigorously stirred for 30minutes. A solid was collected by filtration, washed with water andallowed to dry under reduced pressure to afford the title compound as abeige solid (23.36 g, 52% yield).

LCMS: m/z 397 [M+H]⁺.

Stage2—2-{4-[6-Amino-5-(2,4-difluorobenzoyl)-2-oxopyridin-1(2H)-yl]-3,5-difluorophenyl}ethylacetate

Propiolic acid (5.4 mL, 88 mmol) was added dropwise over 5 minutes to acold (0° C.) solution of CDI (14.27 g, 88 mmol) in anhydrous THF (400mL) under nitrogen. After completion of the addition, the reactionmixture was allowed to warm to room temperature and stirred for onehour. A solution of2-(4-{[3-(2,4-difluorophenyl)-3-oxopropanimidoyl]amino}-3,5-difluoro-phenyl)ethylacetate (23.26 g, 59 mmol) in anhydrous THF (200 mL) was added and thereaction mixture was stirred at reflux for 6.5 hours. The reactionmixture was allowed to cool to room temperature and left standing for16.5 hours. Propiolic acid (5.4 mL, 88 mL), CDI (14.27 g, 88 mmol) andTHF (200 mL) were treated as described above and added to the reactionmixture, which was subsequently stirred at reflux for an additional 6hours. The reaction mixture was then allowed to cool to room temperatureand concentrated under reduced pressure to leave a brown oil.Purification by dry flash chromatography (5% MeOH in DCM) gave a darkbrown solid, which was further purified by trituration with EtOAc toafford the title compound as a yellow solid (7.45 g, 28% yield).

LCMS: m/z 449 [M+H]⁺ and 471 [M+Na]⁺.

Stage3—6-Amino-5-(2,4-difluorobenzoyl)-1-[2,6-difluoro-4-(2-hydroxyethyl)phenyl]pyridin-2(1H)-one

2-{4-[6-Amino-5-(2,4-difluorobenzoyl)-2-oxopyridin-1(2H)-yl]-3,5-difluorophenyl}ethylacetate (7.45 g, 17 mmol) was suspended in 6N HCl (80 mL) and thereaction mixture was refluxed for 21.5 hours. A solid was collected byfiltration, taken up in a saturated aqueous solution of NaHCO₃ (200 mL)and vigorously stirred for 30 minutes. A solid was collected byfiltration, washed with water and dried in a vacuum oven (40° C.) toafford the title compound as a beige solid.

LCMS: m/z 407 [M+H]⁺ and 429 [M+Na]⁺.

¹H NMR (300 MHz, DMSO-d₆) 7.57 (1H, td, J=6.6, 8.3 Hz), 7.41 (1H, td,J=2.4, 9.7 Hz), 7.37-7.29 (3H, m), 7.23 (1H, td, J=2.3, 8.5 Hz), 5.74(1H, d, J=9.8 Hz), 4.78 (1H, t, J=5.1 Hz), 3.76-3.70 (2H, m), 2.86 (2H,t, J=6.7 Hz), —NH ₂ not visible

Stage4—{4-[6-Amino-5-(2,4-difluorobenzoyl)-2-oxopyridin-1(2H)-yl]-3,5-difluorophenyl}-acetaldehyde

Dess-Martin periodinane (1.03 g, 2.4 mmol) was added to a suspension of6-amino-5-(2,4-difluorobenzoyl)-1-[2,6-difluoro-4-(2-hydroxyethyl)phenyl]pyridin-2(1H)-one(823 mg, 2.0 mmol) in DCM (20 mL). The reaction mixture was stirred atroom temperature for 2 hours, quenched with a saturated aqueous solutionof NaHCO₃ (10 mL) and a saturated aqueous solution of sodium thiosulfate(10 mL) and vigorously stirred for 30 minutes. The aqueous layer wasseparated and further extracted with DCM (2×20 mL). The combined organicextracts were dried (MgSO₄), filtered and concentrated under reducedpressure to afford the title compound as a pale brown solid (819 mg).This was used without further purification in the next stage.

Stage 5—tert-ButylN-(2-{4-[6-amino-5-(2,4-difluorobenzoyl)-2-oxopyridin-1(2H)-yl]-3,5-difluorophenyl}ethyl)-L-alaninate

tert-Butyl L-alaninate hydrochloride (552 mg, 3.0 mmol) and STAB (1.29g, 6.1 mmol) were added to a solution of{4-[6-amino-5-(2,4-difluorobenzoyl)-2-oxopyridin-1(2H)-yl]-3,5-difluorophenyl}-acetaldehyde(819 mg, 2.0 mmol). The reaction mixture was stirred at room temperaturefor 3.5 hours, quenched with a saturated aqueous solution of Na₂CO₃ (20mL) and vigorously stirred for 20 minutes. The aqueous layer wasseparated and further extracted with EtOAc (2×20 mL). The combinedorganic extracts were washed with brine (20 mL), dried (MgSO₄), filteredand concentrated under reduced pressure to leave a yellow oil.Purification by column chromatography (5% MeOH in DCM) afforded thetitle compound as a pale yellow solid (492 mg, 78% yield over twosteps).

LCMS: purity 98%, m/z 534 [M+H]⁺.

¹H NMR (300 MHz, DMSO-d₆) 7.58 (1H, td, J=6.8, 8.3 Hz), 7.41 (1H, td,J=2.3, 9.8 Hz), 7.37-7.30 (3H, m), 7.23 (1H, td, J=2.3, 8.5 Hz), 5.74(1H, d, J=9.8 Hz), 3.20 (1H, d, J=7.0 Hz), 2.89-2.70 (4H, m), 1.42 (9H,s), 1.16 (3H, d, J=7.0 Hz), —NH ₂ and —NH— not visible

Measurement of Biological Activities

p38 MAP Kinase Activity

The ability of compounds to inhibit p38 MAP a Kinase activity wasmeasured in an assay performed by Upstate (Dundee UK). In a finalreaction volume of 254 μL, p38 MAP Kinase a (5-10 mU) is incubated with25 mM Tris pH 7.5, 0.002 mMEGTA, 0.33 mg/mL myelin basic protein, 10 mMMgAcetate and [g-33p-ATP] (specific activity approx. 500 cpm/pmol,concentration as required). The reaction is initiated by the addition ofthe MgATP mix. After incubation for 40 minutes at room temperature, thereaction is stopped by the addition of 5 μL of a 3% phosphoric acidsolution. 10 μL of the reaction is then spotted onto a P30 filtermat andwashed three times for 5 minutes in 75 mM phosphoric acid and once inmethanol prior to drying and scintillation counting.

Duplicate data points are generated from a ⅓ log dilution series of astock solution in DMSO. Nine dilutions steps are made from a topconcentration of 10 μM, and a ‘no compound’ blank is included. Thestandard radiometric filter-binding assay is performed at an ATPconcentration at, or close to, the Km. Data from scintillation countsare collected and subjected to free-fit analysis by Prism software. Fromthe curve generated, the concentration giving 50% inhibition isdetermined and reported.

LPS-Stimulation of THP-1 Cells

THP-1 cells were plated in 100 μl at a density of 4×10⁴ cells/well inV-bottomed 96 well tissue culture treated plates and incubated at 37° C.in 5% CO₂ for 16 hrs. 2 hrs after the addition of the inhibitor in 100μl of tissue culture media, the cells were stimulated with LPS (E colistrain 005:B5, Sigma) at a final concentration of 1 μg/ml and incubatedat 37° C. in 5% CO₂ for 6 hrs. TNF-α levels were measured from cell-freesupernatants by sandwich ELISA (R&D Systems #QTA00B).

LPS-Stimulation of Human Whole Blood

Whole blood was taken by venous puncture using heparinised vacutainers(Becton Dickinson) and diluted in an equal volume of RPMI1640 tissueculture media (Sigma). 100 μl was plated in V-bottomed 96 well tissueculture treated plates. 2 hrs after the addition of the inhibitor in 100μl of RPMI1640 media, the blood was stimulated with LPS (E coli strain005:B5, Sigma) at a final concentration of 100 ng/ml and incubated at37° C. in 5% CO₂ for 6 hrs. TNF-α levels were measured from cell-freesupernatants by sandwich ELISA (R&D Systems #QTA00B).

Plasma Exposure in Mice

The compounds were formulated in 8% DMSO, 92% 11.25%hydroxypropyl-β-cyclodextrin in water using the following procedure: thecompounds were fully dissolved in 100% DMSO and then thehydroxypropyl-P-cyclodextrin solution added. The fine precipitate formedwas re-dissolved by the addition of aqueous HCl and the pH adjusted to 4with aqueous sodium hydroxide.

Each compound was administered orally at 10 mg/kg, in a total dosevolume of 5 ml/kg, to male CD1 mice (25-20 g). Three mice were used foreach time point. Blood samples were taken at the following time points:5, 15, 30, 60, 120, 240 and 360 minutes, by terminal cardiac puncture,under halothane/isofluorane anaesthesia. Blood samples were collectedinto pre-chilled tubes containing NaF/EDTA and mixed. Samples were spunat 7-7.5×g for 2 minutes. The plasma was aspirated and frozen.

Plasma samples were prepared by precipitation of protein using threevolumes of acetonitrile containing the internal standard. Thesupernatants were analysed by LCMS (Sciex API 3000, HP1100 binary pump,CTC PAL). The chromatography was based on an Acentis C8 (50×2.1 mm)column and a mobile phase of 5-95% acetonitrile in water/0.1% formicacid.

Exposure (AUC) was calculated from the plasma concentration versus timeprofile using PK Solutions 2.0 (Summit Research Services, Montrose,Colo.).

TABLE 1 Biological Activity of the Compound of the Invention andStructurally Related Compounds Chemical Structure Name A B C D

tert-Butyl N-(2-{4-[6-amino- 5-(2,4-difluorobenzoyl)- 2-oxopyridin-1(2H)-yl]-3,5- difluorophenyl}ethyl)-L- alaninate 12 1 1.6 26

tert-Butyl N-[2-(4-{6- amino-5-[(4-fluorophenyl) carbonyl]-2-oxopyridin-1(2H)- yl}phenyl)ethyl]-L- leucinate 724 118 19.0 947

tert-Butyl N-[2-(4-{6- amino-5-[(2,4- difluorophenyl)carbonyl]-2-oxopyridin- 1(2H)-yl}phenyl)ethyl]- L-leucinate 330 58 2.4638

tert-Butyl (S)-2-(3- {4-[6-Amino- 5-(2,4-difluoro benzoyl)-2-oxo-2H-pyridin-1-yl]- 3,5-difluorophenoxy} propylamino)-4- methylpentanoate 76.1 4274

tert-Butyl N-(5- {4-[6-amino-5- (2,4-difluorobenzoyl)-2-oxopyridin-1(2H)-yl]- 3,5-difluorophenoxy} pentyl)-L-leucinate 73 229.0 1247

Cyclopentyl (S)-(3- {4-[6-Amino- 5-(4-fluorobenzoyl)- 2-oxo-2H-pyridin-1-yl]-3,5- difluorophenoxy} propylamino) phenylacetate 38.0

Cyclopentyl (S)-(3- {4-[6-Amino- 5-(2,4-difluorobenzoyl)-2-oxo-2H-pyridin-1-yl]- 3,5-difluorophenoxy} propylamino) phenyl acetate5.3 508

Cyclopentyl (S)-2-(3-{4-[6- Amino-5-(2,4- difluoro benzoyl)-2-oxo-2H-pyridin-1-yl]- 3,5-difluorophenoxy} propylamino)-4- methylpentanoate 10.7 594

Cyclopentyl (2R)- [(3-{4-[6- amino-5-(2,4- difluorobenzoyl)-2-oxopyridin-1(2H)-yl]-3,5- difluorophenoxy}propyl) amino](phenyl)acetate6.0

2-Morpholin-4- ylethyl N-(3-{4- [6-amino-5-(2,4- difluorobenzoyl)-2-oxopyridin-1(2H)-yl]- 3,5-difluorophenoxy} propyl)-L-leucinate 0.4 935

Cyclopentyl (2S)-4- amino-2-[(3- {4-[6-amino-5-(2,4- difluorobenzoyl)-2-oxopyridin-1(2H)-yl]-3,5- difluorophenoxy}propyl) amino]butanoate 1 522.0 479

Cyclopentyl N-(5- {4-[6-amino-5- (2,4-difluorobenzoyl)-2-oxopyridin-1(2H)- yl]-3,5-difluorophenoxy} pentyl)-L-leucinate 81 24.2 179

Ethyl N-(3-{4-[6-amino-5-(4- fluorobenzoyl)-2- oxopyridin-1(2H)-yl]-3,5- difluorophenoxy} propyl)-L-leucinate 28 8.6

tert-butyl N-[2-(4- {6-amino-5- [(2,4-difluorophenyl) carbonyl]-2-oxopyridin-1(2H)- yl}phenyl)ethyl]-L- alaninate 58 58 2.8 195

tert-butyl N-(2- {4-[6-amino-5- (2,4-difluorobenzoyl)-2-oxopyridin-1(2H)- yl]-3,5-difluorophenyl} ethyl)-L-leucinate 120 1 3.0191

tert-butyl N-(2- {4-[6-amino-5- (2,4-difluorobenzoyl)-2-oxopyridin-1(2H)- yl]-3,5-difluorophenyl} ethyl)-D-leucinate 167 281.0 0% @ 10 μm

cyclopentyl N-(2- {4-[6-amino-5- (2,4-difluorobenzoyl)-2-oxopyridin-1(2H)-yl]-3,5- difluorophenyl} ethyl)-L-leucinate 231 1 0.554

Cyclopentyl N-[2-(4-{6-amino-5- [(2,4-difluorophenyl)carbonyl]-2-oxopyridin- 1(2H)-yl}-3,5 difluorophenyl)ethyl]-L- valinate22 8.4 5184

Cyclopentyl N-[2-(4-{6-amino-5- [(2,4-difluorophenyl)carbonyl]-2-oxopyridin- 1(2H)-yl}phenyl)ethyl]- L-threoninate 68 56 1.81198

Cyclopentyl N-(2-{4-[6-amino-5- (4-fluorobenzoyl)-2-oxopyridin-1(2H)-yl]- 3,5-difluorophenyl} ethyl)-L-leucinate 12 1 0.9190

Cyclopentyl (2S)-[(2-{4-[6- amino-5-(2,4- difluorobenzoyl)-2-oxopyridin-1(2H)-yl]- 3,5-difluorophenyl} ethyl)amino] (phenyl)acetate65 1 0.5 148

tert-butyl (2S)-[(2-{4-[6-amino-5- (2,4-difluorobenzoyl)-2-oxopyridin-1(2H)-yl]-3,5- difluorophenyl}ethyl) amino](phenyl)acetate 441 2.0 779

Cyclopentyl N-(2-{4-[6-amino-5- (2,4-difluorobenzoyl)-2-oxopyridin-1(2H)-yl]-3,5- difluorophenyl}ethyl)- O-tert-butyl-L-serinate57 1 5.7 1295

tert-butyl N-(2-{4-[6-amino-5- (2,4-difluorobenzoyl)-2-oxopyridin-1(2H)-yl]-3,5- difluorophenyl}ethyl)- O-tert-butyl-L-serinate72 1 83.0

Cyclopentyl (2R)-[(2-{4-[6- amino-5-(2,4- difluorobenzoyl)-2-oxopyridin-1(2H)-yl]-3,5- difluorophenyl}ethyl) amino](phenyl)acetate 4721.0 4108 Columns A to D provide data for the following: A—ester enzymeassay (p38 kinase A (invitrogen)), IC50 (nM); B—acid enzyme assay (p38kinase A (invitrogen)), IC50 (nM); C—ester TNF-alpha inhibition (THP-1cells), IC50 (nM); and D—ester TNF-alpha inhibition (human whole blood),IC50 (nM).

TABLE 2 Plasma exposure in mice po 10 mg/kg AUC_(0-t) Chemical StructureName (ng/mL · h)

tert-Butyl N-(2-{4-[6-amino-5- (2,4-difluorobenzoyl)-2-oxopyridin-1(2H)-yl]-3,5-difluorophenyl}ethyl)- L-alaninate 102

tert-butyl N-[2-(4-{6-amino-5-[(2,4- difluorophenyl)carbonyl]-2-oxopyridin-1(2H)-yl} phenyl)ethyl]-L-alaninate 0

tert-butyl N-(2-{4-[6-amino-5-(2,4- difluorobenzoyl)-2-oxopyridin-1(2H)-yl]-3,5- difluorophenyl}ethyl)-L-leucinate 75

cyclopentyl N-(2-{4-[6-amino-5-(2,4- difluorobenzoyl)-2-oxopyridin-1(2H)-yl]-3,5-difluorophenyl} ethyl)-L-leucinate 14

Conclusions

Table 1 demonstrates that the compound of the present inventor is a goodinhibitor of p38 MAP kinase. Further, the IC50 values for ester TNF-αinhibition in THP-1 cells and human blood are very low. In particular,the ester TNF-α inhibition in human blood is significantly lower thanwould be expected given the IC50 values for ester TNF-α inhibition inhuman blood observed for related compounds.

The AUC data presented in Table 2 show that the bioavailability of thecompound of the invention is much higher than that ofstructurally-similar, known compounds.

The invention claimed is:
 1. A method of treating cancer in a subject,which method comprises administering to said subject an effective amountof a compound which is: tert-butylN-[2-{4-[6-amino-5-(2,4-difluorobenzoyl)-2-oxopyridin-1(2H)-yl]-3,5-difluorophenyl}ethyl)-L-alaninate,or a pharmaceutically acceptable salt, hydrate or solvate thereof.
 2. Amethod according to claim 1 wherein the cancer is breast cancer, ovariancancer, pancreatic cancer, lung cancer, colon cancer, renal cancer,lymphoma or melanoma.
 3. A method of treating cancer in a subject, whichmethod comprises administering to said subject an effective amount of apharmaceutical composition comprising a compound, which compound is:tert-butylN-[2-{4-[6-amino-5-(2,4-difluorobenzoyl)-2-oxopyridin-1(2H)-yl]-3,5-difluorophenyl}ethyl)-L-alaninate,or a pharmaceutically acceptable salt, hydrate or solvate thereof, andwhich comprises one or more pharmaceutically acceptable carriers and/orexcipients.
 4. A method according to claim 3, wherein the cancer isbreast cancer, ovarian cancer, pancreatic cancer, lung cancer, coloncancer, renal cancer, lymphoma or melanoma.
 5. A method of treatingcancer in a subject, which method comprises administering to saidsubject an effective amount of: (i) a compound which is apharmaceutically acceptable ethanesulphonate salt of tert-butylN-[2-{4-[6-amino-5-(2,4-difluorobenzoyl)-2-oxopyridin-1(2H)-yl]-3,5-difluorophenyl}ethyl)-L-alaninate;or (ii) a composition comprising a pharmaceutically acceptableethanesulphonate salt of tert- butylN-[2-{4-[6-amino-5-(2,4-difluorobenzoyl)-2-oxopyridin-1(2H)-yl]-3,5-difluorophenyl}ethyl)-L-alaninatetogether with one or more pharmaceutically acceptable carriers and/orexcipients.
 6. A method according to claim 5 wherein the cancer isbreast cancer, ovarian cancer, pancreatic cancer, lung cancer, coloncancer, renal cancer, lymphoma or melanoma.
 7. A method of treatingcancer in a subject, which method comprises administering to saidsubject an effective amount of: (i) a compound which is apharmaceutically acceptable methanesulphonate salt of tert-butylN-[2-{4-[6-amino-5-(2,4-difluorobenzoyl)-2-oxopyridin-1(2H)-yl]-3,5-difluorophenyl}ethyl)-L-alaninate;or (ii) a composition comprising a pharmaceutically acceptablemethanesulphonate salt of tert-butylN-[2-{4-[6-amino-5-(2,4-difluorobenzoyl)-2-oxopyridin-1(2H)-yl]-3,5-difluorophenyl}ethyl)-L-alaninateand comprising one or more pharmaceutically acceptable carriers and/orexcipients.
 8. A method according to claim 7 wherein the cancer isbreast cancer, ovarian cancer, pancreatic cancer, lung cancer, coloncancer, renal cancer, lymphoma or melanoma.
 9. A compound which is apharmaceutically acceptable ethanesulphonate salt of tert-butylN-[2-{4-[6-amino-5-(2,4-difluorobenzoyl)-2-oxopyridin-1(2H)-yl]-3,5-difluorophenyl}ethyl)-L-alaninate.10. A pharmaceutical composition comprising a compound according toclaim 9 and comprising one or more pharmaceutically acceptable carriersand/or excipients.
 11. A compound which is a pharmaceutically acceptablemethanesulphonate salt of tert-butylN-[2-{4-[6-amino-5-(2,4-difluorobenzoyl)-2-oxopyridin-1(2H)-yl]-3,5-difluorophenyl}ethyl)-L-alaninate.12. A pharmaceutical composition comprising a compound according toclaim 11 and comprising one or more pharmaceutically acceptable carriersand/or excipients.